In this project, we seek to fill an important gap in mucosal vaccine delivery. Most pathogens enter the body at mucosal surfaces such as the nasal and vaginal passageways and the intestines, yet nearly all vaccines to date are injected into the muscle or under the skin. These vaccines stimulate a systemic immune response, but often leave the mucosal epithelium, our primary barrier to infection, vulnerable. We propose to develop a novel and adaptable mucosal vaccine platform using T4-bacteriophage capsids. These capsids can simultaneously package vaccine DNA, present protein antigens, and display targeting ligands on the head surface. Therefore, we can engineer a targeted vaccine vector that presents the immune system with a protein-prime and a DNA-boost to generate a long-lasting immune response. To do this, we will target the T4-heads to epithelial cells using the cell-surface receptors toll-like receptor 2 and 5 (TLR2 & 5). These receptors are part of the early innate immune response and are targets of vaccine adjuvants. Triggering an innate immune response is key to developing an adaptive, long-lasting response. We will also optimize a transgene cassette encoding vaccine DNA for high-level and long-term expression in epithelial cells. We hypothesize that by targeting the heads to the epithelium and delivering them to mucosal associated tissues, we will elicit a strong immune response systemically and significantly, in the mucosal epithelium. To test this hypothesis, we will deliver targeted-T4 capsids presenting antigen and carrying vaccine-DNA to mice sublingually (under-the-tongue), intranasally, and intramuscularly. We will then compare the innate and adaptive immune responses at both early (day 3) and late time-points (1 year). We expect to observe the most robust adaptive immune response in the groups with the strongest early innate response. We predict that all routes of administration will generate a systemic response, but we will see the most robust mucosal immune response in the sublingual and the intranasal groups. If successful, the work here will generate an effective, novel, mucosal vaccine platform that is inexpensive to produce and readily adaptable to a multitude of diseases.